Tumor suppressor p53 protein binds to DNA and represses transcriptional activation at the G1-S boundary. Since repressor activity requires translocation of p53 to the nucleus, we are interested in the biochemical events involved in synthesis and regulation of the p53 protein complex.We have shown a number of proteins are bound heat shock proteins (HSP) complexed to p53 while in the cytoplasm but released when the complex enters the nucleus, and our lab has shown there is a family of at least 11 p53 isoforms which originate from the same locus but are post- translationally modified. We have also determined there is a conformational change in both p53 and HSP~s once they enter the nucleus. Since the reasons for complex formation and the biochemical function of the associated proteins which bind to p53 is largely unknown, we plan to isolate and identify the proteins complexed to p53 and study their functional roles in translocation. We have obtained a series of truncated human and rodent p53 constructs missing various segments of the gene including the transactivation and binding domains as well as the oligomerization domain and nuclear localization signals. We will re- construct these genes into compatable mammalian expression vectors containing a neo selection marker. Each construct will have a critical area of the p53 gene absent. These constructs will used be for transfection into human and rodent cells which are null for p53 gene expression with subsequent isolation of p53 and its complex of associated proteins. These constructs will be used to probe the respective roles of p53-complexed proteins in formation and stability of the p53 structure, as well as the mechanism of complex translocation to the nucleus and release of p53 for attachment to DNA.